Abstract
Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.
Highlights
Protein-based affinity reagents offer wideranging applications for basic research and therapeutic approaches
We enhance the T3SS of an avirulent strain of Salmonella enterica serovar typhimurium (S. typhimurium) for effective cytosolic delivery of large amounts of synthetic binding proteins into multiple cell types
The expression of recombinant proteins is under the control of the pSicA promoter and co-regulated with Salmonella pathogenicity island-1 (SPI-1) gene expression
Summary
Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wideranging applications for basic research and therapeutic approaches. In contrast to small molecules, which usually require extensive medicinal chemistry to achieve and demonstrate specificity, recombinant binding proteins such as Designed Ankyrin Repeat Proteins (DARPins)[12], nanobodies[13] or monobodies[14] can readily be selected for high affinities and specificities against almost any target. These macromolecules cannot readily access the cytosolic space which limits their utility for fundamental mechanistic studies and their analytical and therapeutic potential. We show that such delivery allows functional inhibition of RAS signaling
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